Thinking, memory, and motions rely on fast and accurate chemical transmission between neurons, which is mediated by a delicate calcium-triggered membrane fusion process. Malfunctions in this process cause various mental disorders and neurodegenerative diseases. Decades of effort has led to the discovery of highly conserved core machinery that generally drive membrane fusion (SNAREs, or soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and key regulatory proteins that specifically control the synaptic fusion. Recently, major advances have been made that enables the reconstitution of the calcium-dependent membrane fusion in vitro. Nevertheless, this fusion is slow compared with that observed in vivo and the underlying regulatory mechanisms are in debate. As specialized engines for membrane fusion, SNAREs are believed to generate significant force that draws the membranes to close proximity for fusion. Strikingly, the force is produced by progressive folding and assembly of a cognate pair of SNAREs like a zipper. This zippering mechanism also contributes to the specificity of membrane fusion. However, in the tug-of-war between SNAREs and membranes, force may also have profound effects on SNARE assembly and its regulation, which has largely been neglected. Structural and functional studies of SNAREs are facilitated by using the proteins isolated from their membrane environments. But due to lack of their force load, the SNAREs often assemble themselves in an irregular manner that does not correspond to fusion. We hypothesize that force is an indispensible component for functional SNARE assembly and regulation. It can promote SNARE assembly in a correct pathway for fusion and facilitate its regulation. To test this hypothesis, we will provide single SNARE complexes with a controllable force load and detect their functional folding/assembly processes in real time. Using high-resolution optical tweezer force microscopy, we will pinpoint the folding/assembly reaction of these SNAREs at an unprecedented spatiotemporal resolution, molecule-by-molecule and step-by-step. We will measure the accompanying force and energy generation and examine the effects of the opposing force and regulatory proteins upon the assembly process. The novel approach will allow us to directly test the predominant force model for SNARE function. Our research will provide a foundation for understanding the molecular basis of membrane fusion and its regulation and help guide the development of better medicines for a variety of membrane-trafficking-related diseases.